Scientific research plays a fundamental role in the health and development of any society, since all technological advances depend ultimately on scientific discovery and the generation of wealth is intricately dependent on technological advance. Due to their importance, science and technology generally occupy important places in the hierarchical structure of developed societies, and they receive considerable public and private investment. Publicly funded science is almost entirely devoted to discovery, and it is administered and structured in a very similar way throughout the world. Particularly in the biological sciences, this structure, which is very much centered on the individual scientist and his own hypothesis-based investigations, may not be the best suited for either discovery in the context of complex biological systems, or for the efficient advancement of fundamental knowledge into practical utility. The adoption of other organizational paradigms, which permit a more coordinated and interactive research structure, may provide important opportunities to accelerate the scientific process and further enhance its relevance and contribution to society. The key alternative is a structure that incorporates larger organizational units to tackle larger and more complex problems. One example of such a unit is the research network. Brazil has utilized such networks to great effect in genome sequencing projects...
The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH–fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.
The synthesis of virulence factors and other extracellular proteins
responsible for pathogenicity in Staphylococcus aureus is
under the control of the agr locus. A secreted
agr-encoded peptide, AgrD, processed from the AgrD gene
product, is known to be an effector of self-strain activation and
cross-strain inhibition of the agr response. Biochemical
analysis of AgrD peptides isolated from culture supernatants has
suggested that they contain an unusual thiol ester-linked cyclic
structure. In the present work, chemical synthesis is used to confirm
that the mature AgrD peptides contain a thiolactone structure and that
this feature is absolutely necessary for full biological activity. The
AgrD synthetic thiolactone peptides exhibited biological activity
in vivo in a mouse protection test. Structure-activity
studies have allowed key aspects of the peptide structure involved in
the differential activation and inhibition functions to be identified.
Accordingly, we propose a model for activation and inhibition of the
agr response in which the former, but not the latter,
involves specific acylation of the agr transmembrane
Two enzymes, soluble guanylyl cyclase and cytochrome c
oxidase, have been shown to be exquisitely sensitive to nitric
oxide (NO) at low physiological concentrations. Activation of the
soluble guanylyl cyclase by endogenous NO and the
consequent increase in the second messenger cyclic GMP are now known to
control a variety of biological functions. Cytochrome c
oxidase, the terminal enzyme of the mitochondrial respiratory chain, is
inhibited by NO. However, it is not clear whether NO produced by the
constitutive NO synthase interacts with cytochrome c
oxidase, nor is it known what the biological consequences of such an
interaction might be. We now show that NO generated by vascular
endothelial cells under basal and stimulated conditions modulates the
respiration of these cells in response to acute changes in oxygen
concentration. This action occurs at the cytochrome c
oxidase and depends on influx of calcium. Thus, NO plays a
physiological role in adjusting the capacity of this enzyme to use
oxygen, allowing endothelial cells to adapt to acute changes in their
The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity...
Regulation of gene expression through translational control is a fundamental mechanism implicated in many biological processes ranging from memory formation to innate immunity and whose dysregulation contributes to human diseases. Genome wide analyses of translational control strive to identify differential translation independent of cytosolic mRNA levels. For this reason, most studies measure genes’ translation levels as log ratios (translation levels divided by corresponding cytosolic mRNA levels obtained in parallel). Counterintuitively, arising from a mathematical necessity, these log ratios tend to be highly correlated with the cytosolic mRNA levels. Accordingly, they do not effectively correct for cytosolic mRNA level and generate substantial numbers of biological false positives and false negatives. We show that analysis of partial variance, which produces estimates of translational activity that are independent of cytosolic mRNA levels, is a superior alternative. When combined with a variance shrinkage method for estimating error variance, analysis of partial variance has the additional benefit of having greater statistical power and identifying fewer genes as translationally regulated resulting merely from unrealistically low variance estimates rather than from large changes in translational activity. In contrast to log ratios...
The development of optical methods to control cellular functions is important for various biological applications. In particular, heat shock promoter-mediated gene expression systems by laser light are attractive targets for controlling cellular functions. However, previous approaches have considerable technical limitations related to their use of UV, short-wavelength visible (vis), and infrared (IR) laser light, which have poor penetration into biological tissue. Biological tissue is relatively transparent to light inside the diagnostic window at wavelengths of 650–1,100 nm. Here we present a unique optical biotechnological method using carbon nanohorn (CNH) that transforms energy from diagnostic window laser light to heat to control the expression of various genes. We report that with this method, laser irradiation within the diagnostic window resulted in effective heat generation and thus caused heat shock promoter-mediated gene expression. This study provides an important step forward in the development of light-manipulated gene expression technologies.
Articular cartilage repair remains a significant and growing clinical challenge with the aging population. The native extracellular matrix (ECM) of articular cartilage is a 3D structure composed of proteinaceous fibers and a hydrogel ground substance that together provide the physical and biological cues to instruct cell behavior. Here we present fibrous scaffolds composed of poly(vinyl alcohol) and the biological cue chondroitin sulfate with fiber dimensions on the nanoscale for application to articular cartilage repair. The unique, low-density nature of the described nanofiber scaffolds allows for immediate cell infiltration for optimal tissue repair. The capacity for the scaffolds to facilitate cartilage-like tissue formation was evaluated in vitro. Compared with pellet cultures, the nanofiber scaffolds enhance chondrogenic differentiation of mesenchymal stems cells as indicated by increased ECM production and cartilage specific gene expression while also permitting cell proliferation. When implanted into rat osteochondral defects, acellular nanofiber scaffolds supported enhanced chondrogenesis marked by proteoglycan production minimally apparent in defects left empty. Furthermore, inclusion of chondroitin sulfate into the fibers enhanced cartilage-specific type II collagen synthesis in vitro and in vivo. By mimicking physical and biological cues of native ECM...
The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.
Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability.
RNA sequencing of single cells enables measurement of biological variation in heterogeneous cellular populations and dissection of transcriptome complexity that is masked in ensemble measurements of gene expression. The low quantity of RNA in a single cell, however, hinders efficient and consistent reverse transcription and amplification of cDNA, limiting accuracy and obscuring biological variation with high technical noise. We developed a microfluidic approach to prepare cDNA from single cells for high-throughput transcriptome sequencing. The microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise.
Type I interferons (IFNs) are cytokines with important biological effects, including antileukemic and antineoplastic properties. The biological responses of type I IFNs occur via up-transcriptional activation of specific genes, called interferon-stimulated genes (ISGs), ultimately resulting in generation of protein products that mediate their biological effects. In the present study, we provide evidence that the protein S6 kinase 1 Aly/REF-like target (SKAR) plays a critical role in IFN-dependent mRNA translation of ISGs and recruits activated p90 ribosomal protein S6 kinase to the translational machinery. Our data provide evidence for critical and essential roles for SKAR in the generation of type I IFN-dependent antileukemic and antineoplastic responses, underscoring the importance of SKAR functions in IFN signaling.
Many animals use vision to guide their behaviour and to collect relevant information about their environment. The diversity of visual environments and of visually guided tasks has led to a large variety of specialisations of eyes and visual systems. Our knowledge, however, about how the anatomical and physiological properties of eyes and the behavioural strategies of animals relate to the visual signals that are important to them in their natural environment, is extremely limited. In this thesis, I make use of optical, physiological and behavioural analyses to reconstruct the flow of visual information that the fiddler crab Uca vomeris experiences during its daily life on the mudflat. I present a detailed analysis of the first stage of visual processing, the sampling by the ommatidial array of the crabs' compound eye and demonstrate how regional specialisations of optical and sampling resolution reflect the information content and behavioural relevance of different parts of the visual field. Having developed the first intracellular electrophysiological preparation in fiddler crabs, I then examine the spectral sensitivities of photoreceptors - the basis for colour vision. I show that the crabs possess an unusual trichromatic colour vision system featuring a UV-sensitive and a variety of short-wavelength receptor types based on the coexpression of two short-wavelength sensitive pigments. Finally...
The pelagic ocean harbors one of the largest ecosystems on Earth. It is responsible for approximately half of global primary production, sustains worldwide fisheries, and plays an important role in the global carbon cycle. Ocean warming caused by anthropogenic climate change is already starting to impact the marine biota, with possible consequences for ocean productivity and ecosystem services. Because temperature sensitivities of marine autotrophic and heterotrophic processes differ greatly, ocean warming is expected to cause major shifts in the flow of carbon and energy through the pelagic system. Attempts to integrate such biological responses into marine ecosystem and biogeochemical models suffer from a lack of empirical data. Here, we show, using an indoor-mesocosm approach, that rising temperature accelerates respiratory consumption of organic carbon relative to autotrophic production in a natural plankton community. Increasing temperature by 2–6 °C hence decreased the biological drawdown of dissolved inorganic carbon in the surface layer by up to 31%. Moreover, warming shifted the partitioning between particulate and dissolved organic carbon toward an enhanced accumulation of dissolved compounds. In line with these findings...
The current paradigm for tuning synthetic biological systems is through re-engineering system components. Biological systems designed with the inherent ability to be tuned by external stimuli will be more versatile. We engineered Escherichia coli cells to behave as an externally tunable band-pass filter for enzyme activity and small molecules. The band's location can be positioned within a range of 4 orders of magnitude simply by the addition of compounds to the growth medium. Inclusion in the genetic network of an enzyme-substrate pair that functions as an attenuator is a generalizable strategy that enables this tunability. The genetic circuit enabled bacteria growth to be patterned in response to chemical gradients in nonintuitive ways and facilitated the isolation of engineered allosteric enzymes. The application of this strategy to other biological systems will increase their utility for biotechnological applications and their usefulness as a tool for gaining insight into nature's underlying design principles.
Carbon uptake by marine phytoplankton, and its export as organic matter to the ocean interior (i.e., the “biological pump”), lowers the partial pressure of carbon dioxide (pCO2) in the upper ocean and facilitates the diffusive drawdown of atmospheric CO2. Conversely, precipitation of calcium carbonate by marine planktonic calcifiers such as coccolithophorids increases pCO2 and promotes its outgassing (i.e., the “alkalinity pump”). Over the past ≈100 million years, these two carbon fluxes have been modulated by the relative abundance of diatoms and coccolithophores, resulting in biological feedback on atmospheric CO2 and Earth's climate; yet, the processes determining the relative distribution of these two phytoplankton taxa remain poorly understood. We analyzed phytoplankton community composition in the Atlantic Ocean and show that the distribution of diatoms and coccolithophorids is correlated with the nutricline depth, a proxy of nutrient supply to the upper mixed layer of the ocean. Using this analysis in conjunction with a coupled atmosphere–ocean intermediate complexity model, we predict a dramatic reduction in the nutrient supply to the euphotic layer in the coming century as a result of increased thermal stratification. Our findings indicate that...
Astronauts beyond the Earth's orbit are exposed to high-energy cosmic-ray nuclei with high values of linear energy transfer (LET), resulting in much more biological damage than from x-rays or γ-rays and may result in mutations and cancer induction. The relative biological effectiveness of these nuclei depends on the LET, rising to as high as ≈50 at LET values of ≈100-200 keV/μm. An endpoint of concern is germ cell mutations passed on to offspring, arising from exposure to these nuclei. A vertebrate model for germ cell mutation is Medaka fish (Oryzias latipes). We exposed wild type males to doses of 1 GeV per nucleon Fe nuclei or to 290 MeV per nucleon C nuclei. They were mated to females with recessive mutations at five-color loci. The transparent embryos from >100 days of mating (representing exposed sperm, spermatids, or spermatogonia) were observed so as to detect dominant lethal mutations and total color mutations, even though the embryos might not hatch. The relative number of mutant embryos as a function of dose were compared with those induced by γ-rays. The relative biological effectiveness values for dominant lethal mutations and total color mutations for exposed sperm and spermatids were 1.3-2.1 for exposure to C nuclei and 1.5-3.0 for exposure to Fe nuclei. (The spermatogonial data were uncertain.) These low values...
Heterotrimeric G proteins are critical signal-transducing molecules controlled by a complex network of regulators. GIV (a.k.a. Girdin) is a unique component of this network and a nonreceptor guanine nucleotide exchange factor (GEF) that functions via a signature motif. GIV's GEF motif is involved in the regulation of critical biological processes such as phosphoinositide 3 kinase (PI3K)-Akt signaling, actin cytoskeleton remodeling, cell migration, and cancer metastasis. Here we investigated how the GEF function of GIV affects the wiring of its signaling pathway to shape different biological responses. Using a structure-guided approach, we designed a battery of GIV mutants with different Gαi-binding and -activating properties and used it to dissect the specific impact of changes in GIV's GEF activity on several cellular responses. In vivo signaling assays revealed a threshold effect of GEF activity for the activation of Akt by GIV in different cell lines and by different stimuli. Akt signaling is minimal at low GEF activity and is sharply increased to reach a maximum above a threshold of GEF activity, suggesting that GIV is a critical signal amplifier and that activation of Akt is ultrasensitive to changes in GIV's GEF activity. A similar threshold dependence was observed for other biological functions promoted by GIV such as remodeling of the actin cytoskeleton and cell migration. This functional characterization of GIV's GEF motif provides insights into the molecular interactions between nonreceptor GEFs and G proteins and the mechanisms that govern this signal transduction pathway.
Protein O-GlcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GlcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GlcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment–significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GlcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GlcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GlcNAc sites on Bassoon have previously been reported as phosphorylation sites...
Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is ΔU2SHNS...